CN112481373B

CN112481373B - circRNA detection kit for auxiliary diagnosis of autism

Info

Publication number: CN112481373B
Application number: CN202011517202.5A
Authority: CN
Inventors: 崔立华; 杜文冉; 冯福民
Original assignee: North China University of Science and Technology
Current assignee: North China University of Science and Technology
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-01-28
Anticipated expiration: 2040-12-21
Also published as: CN112481373A; ZA202104277B

Abstract

The invention discloses a circRNA detection kit for auxiliary diagnosis of autism, and belongs to the technical field of RNA detection. The kit for auxiliary diagnosis of autism provided by the invention successfully realizes rapid, sensitive and specific detection of the autism marker hsa _ circ _0004566 by extracting total RNA of a sample to be detected, then carrying out reverse transcription of circRNA, and carrying out qPCR on the obtained reverse transcription product so as to detect the relative expression of the target circRNA. The kit for auxiliary diagnosis of the autism provided by the invention can detect the autism marker in a non-invasive (brain tissue) and biological objective manner, can play a role in early identifying an autism child and assisting in diagnosing an autism patient so as to intervene and treat in time, and has great clinical value and social and economic significance.

Description

circRNA detection kit for auxiliary diagnosis of autism

Technical Field

The invention relates to the technical field of RNA detection, in particular to a circRNA detection kit for auxiliary diagnosis of autism.

Background

Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders that seriously affect physical and mental health of children, and the children patients mainly show language disorders, social disorders, narrow interests, stereotypy of behavior, and the like in different degrees. It is mostly ill in infancy. At present, autism becomes the main cause of disability of children under 6 years old in China, seriously harms physical and psychological health of children and brings heavy economic and mental burdens to society and families.

At present, the international method for clinically diagnosing ASD is mainly based on subjective observation and evaluation of speech, behaviors and the like of children patients. Evaluation was performed using the Autism behavioral Scale (ABC), Childhood Autism Rating Scale (CARS), Autism Diagnostic Observation Scale (2 nd edition, ADOS-2), and Autism Diagnostic Interview Scale revision (ADI-R). The information required for diagnosis is basically derived from the description of the sick children by the main care giver of the sick children, so that the information is subjective, a lot of errors are generated, and missed diagnosis and misdiagnosis are easily caused. And for ASD patients without typical symptoms, a long time is needed for accurate identification and diagnosis. This waiting time delays the critical stage of child diagnosis and early intervention, preventing the relief and improvement of the symptoms of autism in children.

ASD is complex in etiology and uncertain in pathogenesis. Although a great deal of research has found genetic factors associated with ASD, these genetic factors can account for only 10% to 30% of the causes of ASD, and no specific ASD causative gene has been found. In recent years, the epigenetic mechanism has been considered as a potential cause of neurodevelopmental abnormalities. The transcripts of 73% of the region in the human genome are non-coding RNAs. Circular RNA is an emerging class of special non-coding RNA molecules that do not have a free 5 'cap and a 3' poly (A) tail, and are covalently bonded to form a circular structure. The circRNA is not easy to be degraded by RNA exonuclease, is more stable than linear RNA, exists in cytoplasm of eukaryotic cells in a large amount, has certain tissue, time sequence and disease specificity, and is a potential biomarker with stable expression.

Accurately determining the epigenetic data of the patient is the premise of accurate medical treatment, and detecting the circRNA related to the autism of the infant suffering from the autism is the necessary premise for effectively diagnosing the autism patient in early stage. However, there is currently no early, objective method of diagnosis for autism. The invention provides a novel, non-invasive, biologically objective circRNA detection kit for the auxiliary diagnosis of autism, and has great social and economic significance and clinical value.

Disclosure of Invention

The invention aims to provide a circRNA detection kit for auxiliary diagnosis of autism, which solves the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides the use of circRNA hsa _ circ _0004566 as a biomarker for autism.

The invention also provides a kit for auxiliary diagnosis of autism, which comprises a primer group for detecting hsa _ circ _ 0004566.

Preferably, the primer set sequence for detecting hsa _ circ _0004566 is shown as SEQ ID NO.1 and SEQ ID NO. 2.

Preferably, the kit further comprises an RNA extraction reagent, Enzyme Mix, Reaction Buffer, Random Primers, nuclease-free water, Premix Ex Taq II and ROX dye II.

Preferably, the kit further comprises a primer group for detecting the internal reference gene.

Preferably, the reference gene is GAPDH, and the primer group sequence for detecting the reference gene is shown as SEQ ID NO.3 and SEQ ID NO. 4.

The invention also provides a method for detecting the circRNA hsa _ circ _0004566 by using the kit for the auxiliary diagnosis of autism, and the using method of the kit is as follows: extracting total RNA of a sample to be detected, then carrying out reverse transcription of circRNA, and carrying out qPCR on the obtained product so as to detect the relative expression of hsa _ circ _ 0004566.

Preferably, the reverse transcription system of the circRNA is: enzyme Mix 2. mu.L, Reaction Buffer, Random Primers 4. mu.L, total RNA500 ng, nuclease-free water make-up 20. mu.L.

Preferably, the qPCR reaction system comprises 10.0 μ L of Premix Ex Taq II and 0.5 μ L of ROX dye II, 0.5 μ L of each of the forward primer and the reverse primer, 2.0 μ L of cDNA2, and 6.5 μ L of nuclease-free water.

Preferably, the qPCR reaction procedure is pre-denaturation at 95 ℃ for 120 s; denaturation at 95 ℃ for 15s and annealing at 63 ℃ for 20s for 50 cycles.

The invention discloses the following technical effects:

the invention provides application of two circRNAs as markers of autism and a kit for auxiliary diagnosis of autism. The kit for auxiliary diagnosis of the autism can detect the autism marker in a non-invasive (brain tissue) and biological objective manner, has the characteristics of rapidness, sensitivity and specificity, can play a role in early identifying an autism child and assisting in diagnosing an autism patient so as to perform timely intervention and treatment, and has great clinical value and social and economic significance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a melting graph of hsa _ circ _ 0004566;

FIG. 2 is a melting curve diagram of GAPDH;

FIG. 3 is a graph comparing the expression levels of hsa _ circ _0004566 in case and control groups;

FIG. 4 is a ROC plot of hsa _ circ _0004566 diagnosis of autism.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

(1) Trizol method for extracting total RNA in blood plasma or blood serum of autism patient

1) Sucking 300 μ L of plasma or serum into a new EP tube, adding 900 μ L of LTrizol, and standing at room temperature for 5 min;

2) adding 300 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 5 min;

3) centrifuging at 12,000rpm at 4 deg.C for 15 min;

4) transferring the supernatant plasma or serum to another clean EP tube (incapable of absorbing oil below the layer), adding 500 μ ul isopropanol, and placing in-20 deg.C refrigerator for 10 min;

5) centrifuging at 12,000pm for 10min at 4 deg.C;

6) the supernatant was discarded and 1ml of pre-cooled 75% CH was added₃CH₂OH；

7) Centrifuging at 7,500mpm at 4 deg.C for 5 min; discarding the supernatant, and air-drying at room temperature for 10-30min (not completely air-drying nor completely air-drying);

8) adding 20 μ L of EPC treated water, and promoting dissolution in 55-60 deg.C water bath for 10 min. All containers and pipettes were enzyme free to prevent RNA degradation.

(2) Agarose gel electrophoresis for RNA quality control

Total RNA purity and concentration were measured using a microplate reader. The OD of each sample was measured at a wavelength of 260mm, and each sample was repeated three times.

The purity (OD) value of each sample ranged from 1.8 to 2.0, and since there were many samples, only the concentration and purity of some of the samples were listed. As shown in table 1.

TABLE 1 concentration and purity of samples

(3) Reverse transcription of circRNA

The total volume of the reverse transcription system was 20. mu.L. The reaction conditions are as follows: 5min at 25 ℃, 60min at 42 ℃ and 5s at 95 ℃. The reagents required for the reaction are shown in table 2.

Reagents used in Table 2

(4) Real-time fluorescent quantitative PCR

qPCR detection primers for hsa _ circ _0004566 were designed by Primer Premier 5.0 software, and the Primer information is shown in Table 3. The total volume of the reaction system was 20. mu.L, and the reagents required for the reaction are shown in Table 4. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 120 s; denaturation at 95 ℃ for 15s, annealing at 63 ℃ for 20s, for 50 cycles, and melting curve detection. The nucleotide sequence of hsa _ circ _0004566 is shown in SEQ ID NO. 5. Melting curves of hsa _ circ _0004566 and GAPDH are shown in FIGS. 1 and 2.

TABLE 3 primer sequence information (SEQ ID NO.1-SEQ ID NO.4)

TABLE 4 reagents used

hsa _ circ _0004566 gene sequence (SEQ ID NO. 5): AAACTCTGTCTCCAAAAAAAAAAAAAAGGCCTTTTAAAAACTTAGCTTACTGGTAATGTTTTGTTTCTGTGGCGAGATTGCCCACTTGGGTTATTTGGTTTCTTACGTATAAAAAGCCCCAGAGCCTCTAAAATCTGGTTTTATGGTGATTTCACAAGATTGTATCAGTGTCATCTACTCTTTTTTGTGCTGGACTTTGGATTTACCTTTAAGTTGTTGCAATTGCTGCATTGTGACGTAAAGGTATTTGATGAATGAAGTTAAGTTTTGGCCTTGGCACAGTGACCAGGAGCTGAGCAGTGGGTCATGGGAGAGGAAGGGCGATGGAATAAACCGTGTGGACCAAATGGAGGGAGACGAGAGCGATGAAAGAACTGAGGCTGAAATTATTATTGACATGAGCTAATCTCACTCTCTTGTTTCAGAATATATACAGCCCTGCTCTGGGACACACCTCCATTGGATTTAAAAGACAGTCCTCGTCAGCACTGACTTTCAGCTATGGAATCGGGTCTCAGTCGCTCGCCCAGGTGGAGTGCAGTGGTGCCACCCTAACTGACTGCAGCCTTGAGCTTCTGTGCGGAAGTGATCCTCCCACCTCGGCCTCTTAAAGTTCTGGGATTATAGGTTTGGCCTAGAGCTCTTTCTTCTTAGGTAACTTTAACTTTTTACCCGTTGATTGTATTAAATAATTTTCTTTCCCTATCCCTAACGTGGCAAATAACCCACTTAAAGGGGTTGATGAGAGTAAGGAGTCCAGAGTGGTAAGAGCATTTAAAACCTCACATTGCTCCACCATTCAGAGAGAGCCGCCATTAGTACCTCCTGGGTTTCCTAACACAGAATGCGTGTAGCCGACTCCTTAGCACCTGGGTTATCATCTTGTCCTGTTGCCATTTCCATAGGTAGAA are provided.

(5) Data processing

Relative expression of the target circ RNA was calculated using the 2- Δ Ct method using GAPDH as the reference gene. Δ Ct ═ Ct (circRNA of interest) -Ct (gapdh).

The relative expression of hsa _ circ _0004566 in the autism case group and the healthy control group were 0.68 ± 0.26 and 1.02 ± 0.21, respectively, with statistical differences (t ═ 6.674, P <0.001), and the case group was down-regulated by 34% compared to the control group, as shown in fig. 3. The results show that there was a significant difference in the expression levels of hsa _ circ _0004566 in the case and control groups.

To assess the potential of hsa _ circ _0004566 as a diagnostic marker for autism, analysis was performed using a receiver operating characteristic curve (ROC). The ROC curve is a comprehensive index reflecting continuous variables of sensitivity and specificity, and is a mutual relation of sensitivity and specificity revealed by a composition method, and a series of sensitivity (SEN in short) and specificity (SPE in short) are calculated by setting the continuous variables into a plurality of different critical values, and then the sensitivity is used as an ordinate, and the (1-specificity) is used as an abscissa to draw a curve. The larger the area under the curve, the higher the diagnostic accuracy. On the ROC curve, the point closest to the top left of the graph is the cut-off value for high sensitivity and specificity. The area under the ROC curve (AUC) is between 1.0 and 0.5. When AUC >0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC has lower accuracy when being 0.5-0.7, AUC has certain accuracy when being 0.7-0.9, and AUC has higher accuracy when being more than 0.9. The Area Under the ROC Curve (AUC) for hsa _ circ _0004566 was 0.803 (95% CI: 0.733-0.872, P <0.001), and the sensitivity and specificity were 82.5% and 76.9%, respectively, see FIG. 4. Therefore, hsa _ circ _0004566 can be used as a reliable and potential biomarker for diagnosing autism, and provides guiding significance for research of autism-related circRNA.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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Claims

1. The application of the primer group for detecting hsa _ circ _0004566 in preparing the detection reagent for the autism biomarker is characterized in that the sequence of the primer group for detecting hsa _ circ _0004566 is shown as SEQ ID No.1 and SEQ ID No. 2.

2. The application of the primer group for detecting hsa _ circ _0004566 in preparing a kit for auxiliary diagnosis of autism is characterized in that the sequence of the primer group for detecting hsa _ circ _0004566 is shown as SEQ ID No.1 and SEQ ID No. 2.

3. The use of claim 2, wherein the kit further comprises an RNA extraction reagent, Enzyme Mix, Reaction Buffer, Random Primers, nuclease-free water, Premix Ex Taq II and ROX dye II.

4. The use of claim 2, wherein the kit further comprises a primer set for detecting an internal reference gene.

5. The use of claim 4, wherein the reference gene is GAPDH, and the primer set sequences for detecting the reference gene are shown as SEQ ID NO.3 and SEQ ID NO. 4.

6. The use according to claim 5, wherein the kit is used in a method comprising: extracting total RNA of a sample to be detected, then carrying out reverse transcription of circRNA, and carrying out qPCR on the obtained product so as to detect the relative expression of hsa _ circ _ 0004566.